Sensitive method for detecting low levels of ATP

ABSTRACT

Methods are provided for sensitive detection of adenosine triphosphate (ATP) in samples using the luciferin-luciferase reaction. Aspects include using a pH composition that maximizes a signal to noise ratio. The maximum signal to noise ratio can be particularly useful with recombinant luciferase including recombinant Coleoptera luciferase.

REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 11/141,076, filed in May 31, 2005, which in turn is acontinuation-in-part of U.S. application Ser. No. 10/843,495, filed May11, 2004, incorporated herein by this reference.

FIELD

The field involved is detection of adenosine triphosphate (ATP) forhygiene monitoring.

BACKGROUND

Determination of cleanliness in industrial, health care and othersettings is important for maintaining good hygiene and sanitation. Forexample, the surfaces of equipment used for food handling, storage orprocessing are major sources of microbial and allergen contamination.Microbial contamination can lead to decreased shelf life of productsand, if pathogens are present, transmission of disease. Similarly,unexpected allergens on food contact surfaces may contaminate food. Suchcontamination has the potential to cause adverse reactions, such as anallergic reaction including hives, anaphylaxis and death, in sensitivepeople who consume or otherwise contact the contaminated food.

Microbial culturing can be used to determine the presence ofmicroorganisms. Culturing, however, is time consuming and, therefore,the necessary “real time” feedback to sanitation and food preparationpersonnel may not be available. As a result, food exposed to surfacesthat are later found to contain potentially harmful microorganisms couldenter the food supply.

During the 1990's various rapid and efficient test methods and deviceswere developed for the detection of contamination on surfaces. Some ofthese methods do not detect microbes directly but instead use markerssuch as adenosine triphosphate (ATP) that are indicative of the presenceof microbes or residual food contamination of a surface. One suchapparatus is the POCKETSWAB-PLUS (POCKETSWAB is a registered trademarkof Charm Sciences, Inc. of Lawrence, Mass.), which rapidly andefficiently detects ATP on surfaces. The POCKETSWAB apparatus detectsATP through the reaction of luciferin and luciferase in the presence ofATP to generate luminescence (light). Luminescence can be measured usinga luminometer. Such ATP detection systems generally provide the userwith an average reading of relative light units (RLU's) over a timeperiod, for example 5 seconds.

Also during the late 1990's, allergen tests were developed to detectallergenic components of foods. These tests are typically in the ELISA(enzyme linked immunosorbent assay) format and require 30 minutes ormore to obtain a result. ELISA allergen tests have generally been moresensitive for detecting allergenic food residues than previouslyavailable ATP tests such as the POCKETSWAB.

Maximizing the sensitivity of ATP detection assays and systems,particularly single service ATP detection assays, could expand theirusefulness. For example, a sensitive ATP detection system could be usedto rapidly screen a surface for food residue at the level of allergentest detection. Regulations require sensitivity of 5 parts per millionpeanut residue for tests that detect peanut allergens.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are schematic views of a sampling test device. FIG. 1Ashows the swab removed from the test device and FIG. 1B shows the swabin a pre-use position within the device.

FIG. 2A shows insertion of a vial into a bench-top luminometer and FIG.2B shows insertion of a complete test device into a hand-heldluminometer.

FIG. 3 graphically shows photon counters per second as a function oftime at various ATP concentrations (0.5, 0.1, 0.01, 0.001 and 0 fmolATP).

FIG. 4 graphically shows photon counters per second as a function oftime at various low ATP concentrations (0.01, 0.001 and 0 fmol ATP).

FIGS. 5A, 5B, 5C and 5D show various curve fits to predict extended RLUcounts from 20 second RLU counts as follows: 5A (Power Curve Fit), 5B(Exponential Curve Fit), 5C (Linear Trend Fit) and 5D (Polynomial CurveFit).

FIG. 6 shows sample data from a Power Curve Fit analysis using twodifferent luminometers at zero fmol ATP and 0.005 fmol ATP.

FIG. 7 shows sample data from a Power Curve Fit analysis using twodifferent luminometers at 0.01 fmol ATP, 0.1 fmol ATP and 1 fmol ATP.

FIG. 8 shows sample data from an Exponential Curve Fit analysis usingtwo different luminometers at zero fmol ATP and 0.005 fmol ATP.

FIG. 9 shows sample data from an Exponential Curve Fit analysis usingtwo different luminometers at 0.01 fmol ATP, 0.1 fmol ATP and 1 fmolATP.

FIG. 10 shows sample data from a Linear Curve Fit analysis using twodifferent luminometers at zero fmol ATP and 0.005 fmol ATP.

FIG. 11 shows sample data from a Linear Curve Fit analysis using twodifferent luminometers at 0.01 fmol ATP, 0.1 fmol ATP and 1 fmol ATP.

FIG. 12 shows sample data from a Polynomial Curve Fit analysis using twodifferent luminometers at zero fmol ATP and 0.005 fmol ATP.

FIG. 13 shows sample data from a Polynomial Curve Fit analysis using twodifferent luminometers at 0.01 fmol ATP, 0.1 fmol ATP and 1 fmol ATP.

FIG. 14 shows sample data from an average of Exponential and Power CurveFit analysis using two different luminometers at zero fmol ATP and 0.005fmol ATP.

FIG. 15 shows sample data from an average of Exponential and Power CurveFit analysis using two different luminometers at 0.01 fmol ATP, 0.1 fmolATP and 1 fmol ATP.

FIG. 16 is a flow chart showing an example of an accumulative algorithmto increase ATP sensitivity of a luminometer using a 20 second count topredict and extended count.

FIGS. 17A and 17B show a flow chart showing an example of an alternativeaccumulative algorithm to increase ATP sensitivity of a luminometerusing a 20 second count to predict and extended count.

SUMMARY

Various aspects involve methods, devices and systems for improveddetection of ATP in a sample. In an embodiment adding a test sample to asolution creates a first admixture and then admixing said firstadmixture with reagents, such as luciferin and luciferase reagents,forms a second admixture. In addition to luciferin and luciferase, saidsecond admixture can include at least one buffer and co-factors, such asmagnesium, for the luciferin-luciferase reaction. The second admixturecan also include one or more detergents. In many cases, the detergent,for example benzalkonium chloride, Triton X-100 or similar suchdetergents alone or in combination, will be provided in the solution towhich the test sample is added to form the first admixture. Ifdetergents are provided, the percentages of each detergent relative tobuffer, can be, for example, in the range of about 0.003% to about 0.1%,for example in the range of 0.003% to about 0.01%, for example 0.005%.The second admixture can also include at least one co-factor for theluciferin-luciferase-ATP reaction, for example magnesium. In an example,the buffer, for example a phosphate buffer including partly, mostly orexclusively dibasic phosphate, is provided at a molar concentration ofless than about 100 millimolar. Other possible buffers include Bis-Trisand Bis-Tris Propane.

The reaction resulting from the creation of the second admixturegenerates luminescence. The luminescence generated is detected and usedas an indication of sample ATP. It will be appreciated that although wegenerally refer to the solution as including the buffer and detergent inliquid form and the reagent, or reagent composition, as including theluciferin-luciferase in solid form, such as in a tablet, it is alsopossible to have luciferin-luciferase in liquid form. Similarly, thebuffer and detergent ingredients can be provided in solid form to berehydrated during, or prior to, testing. Utilizing solid material, forexample in tablet form, that is later hydrated may allow all reagents tobe stored together thereby avoiding the first admixture.

In one aspect, enhanced ATP sensitivity can be achieved when theluciferin-luciferase-ATP reaction occurs at pH other than the pH ofmaximum luminescence output. That pH can change, for example dependingon the type of luciferase used. For example, with certain recombinantluciferase, maximum light output occurs at around pH 7.5 to pH 7.8.However, increased sensitivity can be achieved at pH less than 7.8, forexample about 6.3 to about 7.2, for example about 6.5 to about 7.2 orabout pH 6.5 to about pH 6.9. Such enhanced sensitivity, rather thaninvolving maximum luminescence output, involves maximizing the signal tonoise ratio by decreasing background readings.

Another aspect involves detecting the total light generated from the ATPreaction during a relatively short time, for example about 5 seconds toabout 30 seconds, and extrapolating, for example using a regressionformula, to predict the light that would be generated over a longerperiod of time, for example 5 minutes. The combination of methods fordetection of luminescence and prediction of extended counts, with themethods for enhancing ATP sensitivity using, for example relatively lowpH, optimum buffers and detergents at optimum concentrations can provideincreased ATP sensitivity, for example at the level useful for allergendetection.

Other aspects include using improved detection of ATP as an indicator ofpossible allergen contamination or as an indicator of relatively lowlevels of microbial contamination.

Another aspect includes an easy to use all-in-one device for ATPdetection at improved sensitivity levels. Such a device can include alongitudinal housing having a one end and another end and a moveableprobe within the housing to collect a test sample. A transparent closedbottom end can extend from the one end of the housing for use to detectthe luminescence generated from a test sample of a variety of possiblevolumes, for example 300 microliters. In an of an all-in-one device amoveable probe is arranged to puncture at least one membrane seal whichseparates various mixtures, for example a solution from a reagentcomposition. In another example, the membrane seals surround a chamberin which a solution, or, alternatively, a reagent composition, can beprovided.

Another aspect includes improved hygiene monitoring through improvedmethods for measuring and interpreting luminescence output. In one suchmethod, a sample is added to reagents, such as luciferin-luciferase,that generates luminescence in the presence of ATP. The sample andreagents are combined in a container that is inserted into aluminescence detector, such as a photomultiplier based detector, aphotodiode based detector or other detectors capable of measuringluminescence. The luminescence detector detects, quantifies, and storesin memory, the total luminescence output from the sample and reagents,with or without the background subtracted, during a predetermined periodof time. Total luminescence generated is detected, for example bydetecting RLU's generated per second and adding together the total RLU'sgenerated, for example, during a time period of about 5 seconds to about60 seconds or more, for example about 20 seconds. That total can be usedas the result or, alternatively, used to predict the total luminescencethat would be generated during a longer period of time, for exampleduring 5 minutes. By using a shorter count, for example, a 20 secondcount, to accurately extrapolate (predict) a longer count, such as a 5minute count, sensitivity can be increased. The program used forcalculating or predicting total RLU's can be internal to theluminescence detector or external, for example in an accessory computer.

To predict the RLU's that would be generated during, for example, a 5minute count, a variety of possible formulas can be used including alinear or non-linear regression formula. Examples of such non-linearregression formulas include power curve formulas, exponential curveformulas and polynomial curve formulas. In addition, it is possible topredict using the average results from two or more formulas.

The described method for using RLU totals, rather than RLU averages, andpredicting extended time RLU totals from shorter time RLU totals, can beused in combination with the herein described pH conditions and/orreagents, such as the particular types of buffers and/or detergents atthe relevant concentrations. Using such combinations of RLU totaling canincrease ATP sensitivity to the level required for indication ofallergen potential.

Examples of potential applications for the herein described sensitiveATP detection methods, devices and systems include testing water, suchas spring water or other bottled water, for contamination and testingwater to be used by pharmaceutical companies to meet USPC standards.Another possible application is in dairies that share production linesbetween dairy and non-dairy items and where dairy residue in thenon-dairy materials is an allergenic concern. Still another applicationis as an indirect method for screening a surface for allergenic foods; apositive result would tell the user that food residue ATP may exist atthe level of allergen test detection. Another potential applicationincludes a test to detect the potential for contamination on surfacesor, for example, in drinking water. In one embodiment, a sample, forexample a water sample, is filtered to concentrate organic matter andthen the concentrate is tested for the presence of ATP.

Another aspect is a screening test that can be used alone or in atesting system in combination with a confirmatory specific allergendetection test, such as an antibody-binding assay, for detection ofspecific allergens such as peanut allergen. Food residue related ATPthat may be detected using this method include, without limitation,peanut, soy nut, peanut butter, almond, walnut, pecan, egg white, wholeegg, pasteurized whole milk, whole wheat flour, white flour, raw clams,raw shrimp, salmon, sunflower seeds, sesame seeds, powdered milk, soyflour and ultra high temperature milk.

Use of such an ATP detection method for allergen detection, alone or incombination with specific allergen tests, can reduce testing costs andtesting time by allowing rapid, inexpensive surface screening. Such atest would also be useful in expediting remediation of suspect surfaces.After surface cleanliness has been determined with the ATP test system,confirmation of either lack of, or presence of, specific allergen can bemade with a specific allergen test.

Definitions

Within this application we use the term “buffer” both in accordance withits ordinary meaning and with an additional meaning. The ordinarymeaning in the art is a solution that resists change in pH when acid oralkali is added. We also use the term “buffer” to refer to solutions, orother organizations of material including solids, powder, and tabletsthat may be later reconstituted.

DETAILED DESCRIPTION

Embodiments include a method for optimizing or increasing thesensitivity of ATP detection systems. The method is useful as atechnique to increase ATP sensitivity of an ATP detection test device orsystem independent of the concentrations of costly components such asluciferin or luciferase. In addition to increasing test costs,increasing the concentrations of luciferin and/or luciferase canincrease the background of the test system. Increasing the backgroundcan cause misleading results by decreasing the signal to noise ratio.With high background, or noise, a negative sample may generate a readingunrelated to the amount of contamination detected and, thereby,decreasing the efficiency of the system.

In some embodiments the ATP detection method involves a test device witha foam tip, or other absorbent type swab or wand for sample uptake fromthe surface to be monitored. The swab can be pre-moistened with awetting solution, for example, with the same solution used elsewhere inthe system such as a buffering-ATP releasing solution (BAR solution).After sample uptake (for example by absorption through swabbing asurface, pipetting onto the swab or dipping the swab into a sample) ontoa swab, the swab is used to contact the sample with the variouscomponents of the device. In an embodiment, the swab first contacts aBAR solution and then contacts a luciferin/luciferase reagentcomposition.

In one embodiment, luciferin is purified beetle D-Luciferin free acidfrom Regis Technologies, Inc., Catalog # 360100 andrecombinant-luciferase (r-luciferase) is from PROMEGA (cloned gene fromPhotinus pyralis) (Promega is a registered trademark of PromegaCorporation, Madison, Wis.). In an example, luciferase had a specificactivity of 3.9×10¹⁰ relative light units per mg protein (sample minimumspecification of 2.0×10¹⁰ relative light units per mg protein). Althoughmany specific examples described herein include the above describedrecombinant luciferase from PROMEGA a variety of natural and recombinantluciferases are known in the art and can be usefully employed in variousaspects and embodiments including those described in U.S. Patentapplication number 2005/0079567 A1, Choi et al., published Apr. 14,2005; U.S. Pat. No. 6,812,012, Hattori et al., issued Nov. 2, 2004; U.S.Pat. No. 6,265,177 B1, Squirrell et al., issued Jul. 24, 2001; U.S. Pat.No. 5,744,320, Sherf et al., issued Apr. 29, 1998; and U.S. Pat. No.5,583,024, McElroy et al., issued Dec. 10, 1996, the teachings of allbeing incorporated herein by this reference. For example, in oneembodiment of the disclosure, the luciferase may be a recombinantexpression of Coleoptera luciferase as presented in McElroy et al.,wherein the Coleoptera luciferase catalyzes the oxidation of Coleopteraluciferin to yield light. The Coleoptera luciferase may be produced byexpressing in a prokaryotic or eukaryotic cell, a lysate of said cell ora cell-free protein translation system a recombinant DNA coding forColeoptera luciferase, wherein said Coleoptera luciferase catalyzes theoxidation of Coleoptera luciferin to yield light.

Luciferin and luciferase can be freeze dried together, for example withATP-free bulking agents and stabilizers or provided separately for latermixing. The reagents can be provided in a tablet or not tableted.Although the ratio of luciferin and/or luciferase to BAR solution canvary, exemplary ratios include a ratio of about 0.07 to about 0.08micrograms luciferin per microliter BAR solution and about 0.007 toabout 0.008 micrograms luciferase per microliter BAR solution. In aspecific example, 300 microliters of BAR solution were used. The ratioof luciferase and luciferin can be adjusted to achieve optimum results.For example, the ratio of luciferase to BAR solution can be increasedalong with, or independently of, increasing the ratio of luciferin toBAR solution. The ratio can also be adjusted in combination with otheroptimization methods, such as using a regression formula, to achieveoptimum results. It is also possible, utilizing the improved methods ofreading the luminescence output described herein, to reduce the ratio oramount of luciferase and/or luciferin thereby decreasing the cost pertest and the test background.

In an embodiment in which ATP sensitivity is increased withoutincreasing luciferin or luciferase concentrations, the BAR solution isbuffer, such as Bis-Tris or phosphate buffer such as a combination ofpurified water and potassium phosphate, for example, dibasic potassiumphosphate. The molarity of the buffer can be adjusted to optimize testsensitivity while providing sufficient buffering capacity to maintainthe desired pH of the reaction. For example, concentrations of buffersin the range of about 0.1 millimolar to about 10 millimolar can increasetest sensitivity. Buffering capacity, however, will be minimal. Higherconcentrations, for example in the range of about 25 millimolar to about100 millimolar, can also be used to increase the buffering capacitywhile maintaining high sensitivity. The pH of the buffer can be adjustedto the desired range using, for example, sodium hydroxide or phosphoricacid. One useful phosphate buffer, such as when the sample does notrequire a lot of buffering for the desired pH range, is known asButterfield's Buffer. Another useful phosphate buffer utilizes dibasicphosphate and can be combined with a detergent, such as benzalkoniumchloride, Triton X-100 or the like. Other useful phosphate buffersinclude various mono, di and polyphosphates and their various salts forexample phosphoric acid and its sodium, potassium or other salts such asmonobasic sodium phosphate, dibasic sodium phosphate, tribasic sodiumphosphate, pyrophosphoric acid and other salts and polyphosphoric acidand its various salts, Bis-Tris and Bis-Tris Propane, all of which canalso be combined with detergents.

Detergents can be included in the BAR solution. Examples of BARsolutions that may be improved with detergents include phosphate buffer,Bis-Tris buffer and Bis-Tris propane buffer. Possible detergents orcombinations of detergents are known to those skilled in the art andinclude nonionic detergents such as Triton X-100, Tween 20, Tween 80,Nonidet P40 and n-Undecyl Beta-D glucopyranoside; zwitterionicdetergents such as n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; and cationic detergents such asalkyltrimethylethylammonium bromides, cetyldimethylethylammoniumbromide, dodecyltrimethylammonium bromide, and cetyltrimethylammoniumbromide. The concentration of detergent solution varies for each type ofdetergent. In one embodiment a detergent, for example a quaternaryammonium compound such as benzalkonium chloride, is added to the buffersolution. In an embodiment, Triton X-100 is also included. The one ormore detergents can each alone or combined have a concentration in thebuffer of less than about 0.1%, for example less than about 0.03%. In aspecific example, the concentration of benzalkonium chloride was about0.02% and the concentration of Triton X-100 was about 0.05% in a 50 mMBis-Tris buffer, pH 6.6. Such detergents at such concentrations canimprove test sensitivity.

In a particular embodiment, the BAR solution includes a combination ofabout 10 millimolar dibasic phosphate buffer combined with about 0.005%benzalkonium chloride. In another embodiment, the BAR solution includesless than about 1.0 millimolar dibasic phosphate buffer, for exampleabout 0.1 to about 0.5 millimolar dibasic phosphate buffer. In aparticular embodiment, about 0.2 to about 0.3 millimolar dibasicphosphate buffer is combined with benzalkonium chloride in a ratio ofabout 99.995% phosphate buffer to about 0.005% benzalkonium chloride, toa total volume of about 300 microliters.

In an example using increased luciferin and/or increased luciferaserelative to BAR solution as a method of increasing test sensitivity toATP, a BAR solution of 3.138% Trizma Base, 3.125% phosphoric aciddetergent, 1.344% Tricine, 1.344% Triton X-100 (10% solution) and 0.172%benzalkonium chloride (10% solution) and deionized water was prepared.(Displacement measurements for Trizma base and tricine were used tocalculate the volume of deionized water needed.) The molarity of the BARsolution as described above, is greater than about 300 millimolar. Theratio of luciferase to BAR solution was 0.2409 micrograms luciferase permicroliter BAR solution and the ratio of luciferin to BAR solution was0.481 micrograms luciferin per microliter BAR solution. The result,using a five second non-cumulative (average) RLU count was a 100 foldincrease in assay sensitivity, as compared with a similar formulationutilizing the Charm POCKETSWAB Plus (POCKETSWAB Plus containingapproximately 0.07 luciferin to BAR solution and 0.007 luciferase to BARsolution), allowing detection of 0.05 femtomoles (fmoles) ATP. It willbe appreciated that these formulations could also be used with thevarious improved RLU counting and calculating methods described herein.

An embodiment, using a decrease in luciferase concentration, as comparedto the above example and a five second non-cumulative (average) RLUcount, demonstrates that by decreasing the molarity of the BAR solutionATP sensitivity may be increased. In this embodiment, BAR solution waswater of about pH 6. The ratio of luciferase to BAR solution was 0.0365micrograms per microliter (less than the 0.2409 micrograms permicroliter described in the previous example) and the ratio of luciferinto BAR was about 0.07 micrograms per microliter. The result was a 100fold increase in assay sensitivity relative to Charm POCKETSWAB Plus(POCKETSWAB Plus containing approximately 0.07 luciferin to BAR solutionand 0.007 luciferase to BAR solution), allowing a maximum sensitivity ofapproximately 0.05 femtomoles ATP. Peanut butter was detectable with asensitivity of 5 parts per million. This formulation can also be usedwith the various improved RLU counting and calculating methods describedherein.

Other embodiments include using low molarity BAR solutions as a methodof increasing test sensitivity to ATP (increasing luminescence output)without increasing luciferin/luciferase ratios relative to BAR solutionand with or without the various improved RLU counting and calculatingmethods described herein. Specific embodiments for providing an increasein ATP sensitivity without relying on changes in luciferin/luciferaseratios relative to BAR solution include utilizing buffers useful in thepH range of about 6.3 to about 7.2, such as phosphate buffer, Bis-Tris,Bis-Tris Propane and the like, alone or in combination with non-ionic orionic detergents such as benzalkonium chloride and/or Triton X-100 forexample with individual detergent concentrations below about 0.5%. Thefollowing are examples of possible specific BAR solutions that may beuseful; 1) phosphate buffer, for example Butterfield's Buffer (less than1 millimolar dibasic phosphate); 2) phosphate buffer, for exampleButterfield's Buffer containing a detergent, such as a quaternaryammonium compound such as benzalkonium chloride, for example in aconcentration less than 0.1%; 3) water pH of about 5.5 to pH of about6.5; 4) water pH about 5.5 to about 6.5 with detergent such as aquaternary ammonium compound, such as benzalkonium chloride; 5)Tris-tricine buffer; 6) dibasic phosphate buffer; 7) dibasic phosphatebuffer, containing a detergent, such as a quaternary ammonium compound,such as benzalkonium chloride, for example in a concentration less than0.1% with a pH less than about pH 7.2; 8) tricine buffer, for exampleabout 10 millimolar to about 100 millimolar tricine with or without adetergent such as quaternary ammonium compound, such as benzalkoniumchloride, for example in a concentration less than 0.1%; and 9) dibasicphosphate buffer, such as Butterfield's Buffer or equivalent, forexample with a molarity of about 0.2 mM to about 0.5 mM with or withoutdetergent such as benzalkonium chloride, for example in a ratio of about99.995% phosphate buffer to about 0.005% benzalkonium chloride, with apH of about 6.9; 10) Bis-Tris buffer of molarity less than about 100 mMsuch as about 50 mM with or without detergents such as benzalkoniumchloride and/or Triton X-100 or the like and with exemplary individualdetergent concentrations below about 0.5% for non-ionic detergents suchas Triton X-100 and below about 0.1% for ionic detergents such asbenzalkonium chloride; 11) phosphate buffer of molarity less than about100 mM such as about 50 mM with or without detergents such asbenzalkonium chloride and/or Triton X-100 or the like with exemplaryindividual detergent concentrations below about 0.5% for non-ionicdetergents such as Triton X-100 and below about 0.1% for ionicdetergents such as benzalkonium chloride.

In one embodiment, using the BAR solution described in 9 above, the pHof the BAR solution is less than about 7.2, approximately pH 6.9. TheBAR solution was combined with a luciferin and luciferase reagentcomposition including other buffers, co-factors and stabilizers wellknown in the art. Approximately 0.24 millimolar dibasic potassiumphosphate was used in the BAR solution and, approximately, 0.03micrograms glycine (1.4 millimolar when combined with 300 microliter BARsolution) was used in the luciferin and luciferase reagent composition.In such an embodiment, the final reaction pH will be, for example lessthan about 6.9 and the molar concentration of total buffer, includingphosphate and glycine will be less than about 2 millimolar.

In another embodiment the BAR solution was provided within a chamber ofa test system, said chamber sealed with at least one, and generally two,puncturable membrane seals. Examples of test systems utilizing suchchambers (herein sometimes referred to as niblets) include thePOCKETSWAB-Plus, POCKETSWAB Ultra, POCKETH2O, ALLERGIENE, WATERGIENE(Charm Sciences, Inc. Lawrence, Mass.). In some embodiments such testsystems may be brought to room temperature prior to use. After samplecontact, the swab can be used to puncture the niblet membrane, ormembrane seals of a series of niblets, thereby releasing test reagentsinto a test vial and activating the necessary reagents. Generally, thetest vial is a transparent or translucent vial, which permits thepassage or emission of generated luminescence, for example, in abioluminescent assay, and, for example, permits luminescencetransmission of from about 300 to 650 nanometers, which is the visiblelight range. When other reagents within the test vial are dry, such asin tablet or powder form, a desiccant can also be provided within thetest vial such as molecular sieve 4×8 mesh desiccant (AGM ContainerControls, Inc. Tucson, Ariz.)

Methods and test devices for luminescence based ATP detection requirereaders, such as photomultiplier based readers generally known asluminometers. Examples of such luminometers include those described inU.S. Pat. No. 6,055,050. The luminometer may be used in combination witha system including reagents for generation of luminescence in thepresence of ATP, for example the single service ATP detection deviceknown as the POCKETSWAB and described in U.S. Pat. No. 6,055,050 andfurther described in U.S. Pat. No. 5,965,453 (Test Apparatus, System andMethod for the Detection of Test Samples); U.S. Pat. No. 5,985,675 (TestDevice for Detection of an Analyte) and U.S. Reissue patent applicationSer. No. 10/014,154; U.S. Pat. No. 6,180,395 (Reagent Chamber for TestApparatus and Test Apparatus); all of which are incorporated herein bythis reference. The luminescence reader may, for example, be in theformat of the LUMINATOR-K, LUMINATOR-T, FIREFLY, LUM-96 and NOVALUMreaders (Luminator, Firefly, LUM-96 and NovaLum are trademarks of CharmSciences, Inc.; Lawrence, Mass.) The luminescence reader may also be inthe format of any luminescence reading device that detects RLU's such asby using a photodiode, or as with a photomultiplier based luminometer.In these embodiments, the test apparatus provides a user with theluminescence emission count, in RLU'S, of a test sample.

In some embodiments the test result is compared with a background. Whenusing an RLU reading as a measure of sample ATP one possible source ofbackground counts is electrical noise. In certain light detectionsystems such as photomultiplier based systems, a source of backgroundcounts may be what is known in the art as “dark counts” resulting from,for example, thermal, chemiluminescent, or fluorescent emissions fromtest components. It is also possible that background counts result fromoutside light sources if the light detection mechanism is not containedwithin a tightly light sealed environment. In well-designed andconstructed equipment, such sources of background counts are relativelyminimal.

In some embodiments the system includes using a black swab includingblack swab shaft and/or absorbent tip. Such black swab can be used toreduce the amount of light that is absorbed and/or reflected by thesystem that does not relate to ATP luminescence from the sample. Anothersource of background counts is residual ATP present in test reagents. Amethod for eliminating the impact of background counts is to program thereader to remove background counts from the result. When backgroundcounts are deducted, sensitivity of the system is reflected by thesignal to noise ratio rather than the total luminescence.

In several embodiments a background is determined for a particularinstrument by running multiple tests without sample. In an example, aPOCKETSWAB, for example a POCKETSWAB with BAR solution andluciferin/luciferase concentration chosen to be adequately sensitive andcost effective is not contacted with a sample and is instead contactedwith only the reagents and solutions of the system and counted on aluminometer (“activated negative swab”). In one particular example 30activated negative swabs are counted with a 5 minute cumulative count(300 RLU counts per second counts added together). The standarddeviation of the counts from 30 negative swabs is determined and used toadjust the background reading to assure a positive test result is notcaused by a high background reading. In another example, a certainnumber of standard deviations, from about 2 standard deviations to about5 or more, or fractions thereof, are added to the median or averagecounts. For example, in one embodiment, 2.5 standard deviations areadded to the median result of the multiple, for example, 30 readings. Inanother example, 3 standard deviations are added. The standard deviationadjustment will vary and is at least partially dependent on reagentconsistency, and target test specificity and sensitivity.

In another embodiment, 30 activated negative swabs are read on multipleluminometers. The background is set relative to counts of the activatednegative swabs on the multiple luminometers.

After calculation, the background can be programmed into a reader. As asample count proceeds the reader program can compare cumulative readingsto the background. In an embodiment, if the cumulative reading becomesgreater than the background, then the sample is determined to containATP. The background of the reader can be set so that counts above zeroindicate a positive result. In another embodiment, counts above a valueother than zero indicate a positive result.

Cumulative readings can exceed background at any time up to the maximumpredetermined count time, for example 5 minutes. If the maximumpredetermined count time is reached, and the cumulative readings havenot exceeded the background, the sample is negative. Conversely, forhighly contaminated surfaces, cumulative readings can exceed backgroundrelatively quickly making it unnecessary to continue with the full 5minute count.

In another embodiment, the luminometer, for example a luminometerdescribed above, can be optimized by adjusting the luminescence outputreading and interpretation, for example by using a cumulative RLU/secondreading, a peak RLU reading and/or an integrating RLU/second reading.These readings can be over 5 seconds or, to increase test sensitivity,over an extended period of time such as 10 seconds, 20 seconds or 30seconds up to 5 minutes or more. This method for optimizing theluminometer can be used alone or in combination with increasingluciferin/luciferase levels and/or improved BAR solutions, as a methodfor increasing test sensitivity. In addition to the use withphotomultiplier based luminometers, or other luminescence outputreaders, such methods for increasing test sensitivity can be used withother types of light detectors such as photodiodes.

In some embodiments, after contacting the sample with the BAR solutionto create a first admixture, the first admixture is contacted withluciferin and luciferase reagents to create a second admixture. Thereaction of ATP from the sample with the luciferin/luciferase reagentsgenerates luminescence that is detected by a reader. The reader detectsRLU's, for example RLU's emitted per second. Said reader can beprogrammed to detect RLU's over a period of time ranging from a fewseconds, for example 5 seconds, to several minutes, for example 5 or 10minutes. It will be appreciated by those skilled in the art that areading of RLU/second is common in the ATP hygiene monitoring industry.It is possible, however, to change the RLU/second to a different RLU pertime reading, for example, RLU per one-half second or RLU per 2 seconds.For example, in a 5 minute count, the reader can take a count of theRLU's emitted from the sample every second generating 300 readings. Thetotal of those 300 readings can be the test result.

In one embodiment, desired ATP sensitivity levels are achieved throughoptimizing the light output reading and interpretation, for example byusing a cumulative or integrated RLU/second reading or a peak RLUreading, over an extended period of time alone or in combination withincreasing luciferin/luciferase levels and improving the BAR solution.

Other possible examples include using a reader, or a connected CPU,programmed to: 1) take 300 readings over the course of 5 minutes—ofthose 300 readings a subset is chosen, for example a subset defined bythe highest 100 readings which are accumulated to provide a test result;2) take 300 readings and determine the highest, or peak, reading andchose a subset of readings chosen by reference to the peak, for example50 readings prior and 50 readings subsequent to the peak, as the subsetto accumulate readings; 3) integrate the counts and the result reflectsthe area under the integration curve; 4) take the median reading andchoose a subset of readings, by reference to the median reading, to usein the result calculation; 5) determine the peak RLU/second reading andusing that peak reading as the test result; 6) use a group of peakreadings, for example the highest 50 readings to generate the peakreading result and average, accumulate or otherwise manipulate thosemedian reading to arrive at the “peak” readings; and 7) calculate therate of change of the RLU/second readings over a given period of timeand use that rate of change to determine the test result.

In another embodiment the rate of change of the RLU/second (counts persecond) readings is determined over a given period of time. That rate ofchange is used to determine the test result, for example by a softwareprogram within the reader or a software program in a separate computerto which reader results are downloaded.

The method of reading results described herein, and the various BARsolutions, can be used with a variety of luminescence generatingreagents including chemiluminescent reagents, for example dioxetanederivatives such as those available from APPLIED BIOSYSTEMS (AppliedBiosystems is a registered trademark of Perkin-Elmer Corporation, FosterCity, Calif.) and bioluminescent reagents, for example those availablefrom PROMEGA. The method can also be used with a variety of luciferasesdescribed herein from both natural and recombinant sources includingheat and/or detergent stable recombinant luciferase. The insectluciferin can be derived from bioluminescent insects, for exampleDiptera and Coleoptera. Other insect species that display the phenomenaof bioluminescence can also be suitably employed. Including, forexample, species from the orders Diptera and Coleoptera, including thefamilies Lampyridae (including genus Photinus) and Elateridae. Using,for example dioxetane derivatives, compounds other than ATP can bedetected and used, for example, as markers of contamination.

As described above, we have found that extending the count time beyond 5seconds, for example to 5 minutes, and accumulating the counts, ratherthan averaging the counts, provides increased sensitivity as compared toa 5 second average RLU count. It also may be desirable to provide a morerapid result to the user. It might not be practical or desirable to waitone or more minutes for results. In embodiments, results similar tothose observed in an extended cumulative count are calculated fromcounts of a shorter time period, for example counting RLU's per second,for about 5 seconds to about 30 seconds, adding together the RLU persecond counts to arrive at the total RLU generated during those times,and then using one of a variety of possible formulas, to predict totalcounts over an extended longer time. These methods can be used with avariety of ATP detection methods and reagent formulations for examplethose described heretofore and hereinafter within this application. Inthese embodiments, sample RLU results, after a brief period of time, forexample 5 seconds, 10 seconds, 20 seconds, or 30 seconds, are used tocalculate an expected result after, for example 5 minutes. In oneexample, a regression curve formula is used to calculate an expected 5minute result from an actual 20 second cumulative RLU result. Examplesof useful regression formulas include power curve, exponential curve,polynomial curve and linear trend formulas. Some specific examplesinclude:

1. A power curve method in which y=1.2539*x^(1.183) where y is theresult sought (the predicted result after 5 minutes) and x is the 20second result converted to counts per minute and corrected by referenceto a standard LUMINATOR-T.2. An exponential curve method in which y=59597*e^((3.3426E-05x)) wherey is the result sought (the predicted result after 5 minutes) and x isthe 20 second result converted to counts per minute and corrected byreference to a standard LUMINATOR-T.3. A linear equation method in which y=60594+11.261x where y is theresult sought (the predicted result after 5 minutes) and x is the 20second result converted to counts per minute and corrected by referenceto a standard LUMINATOR-T.4. A polynomial curve method in which y=−13737+7.5027*x+3.9039E-05x²where y is the result sought (the predicted result after 5 minutes) andx is the 20 second result converted to counts per minute and correctedby reference to a standard LUMINATOR-T

In another example, results from two or more regression formulas areaveraged to arrive at a result. For example, results from a givenpolynomial curve formula and results from a given power curve formulacan be averaged to provide a final predicted 5 minute reading.

It will be appreciated that the program for manipulating, interpretingor calculating results, using one or more types of regression analysis,will be internal to the particular reader, for example luminometer orphotodiode based reader, or contained in a separate system to which theparticular reader results are downloaded or otherwise accessible. Itwill also be appreciated that there are many derivations of the abovespecific equations and that the above equations are provided by way ofexample only.

Prediction of a 300 second, or other extended time counts, from ashorter time count, can also be made using a variable time assay. Such avariable time assay can be useful if RLU counts are increasing during,for example, the first 20 second count. If the counts are increasingmore then predicted the RLU may be lower than what the actual might havebeen if the full, for example, 300 one second counts were taken. Toreduce or eliminate this problem the RLU count can be summed over a timeframe until the counts obtained do not vary significantly from theaverage of the accumulated sum. For example, the RLU count can beaccumulated over 20 seconds and then a determination can be made by thesoftware program whether or not the RLU count is significantlyincreasing from either the average summed RLU count or the RLU count atthe 20th second. If, on the one hand, the additional RLU count, forexample from the count at the 20th second, is less than a predefinedpercentage, for example 20%, of the average of the accumulated RLUcounts, then the summed RLU count at 20 seconds is used to estimate theRLU count at 300 seconds. If, on the other hand, the RLU count at the20th second is greater than a predefined percentage, for example, 20%,of the average of accumulated RLU counts at 20 seconds, then additionalRLU counts are accumulated until the RLU count at any given second isless than the average of the summed RLU counts up to that time. Thetotal RLU counts summed at that time are then used to estimate theextended count RLU, for example the projected 300 second count.

Another method for projecting longer time period RLU counts from shortertime period counts is using a lookup table. For example, if RLU countsare increasing during the first 20 seconds then the correction topredict the 300 second RLU count is greater than that used when the RLUcount is decreasing toward the end of the 20 second count. For example,if the RLU count is 3000 at 20 seconds, and the curve is decreasing,then multiply the 20^(th) second count by 20. If the RLU count is 10000,and the curve is increasing, then the highest point on the RLU curve hasnot been reached. The 20^(th) second count would, therefore, bemultiplied by a higher number, for example 50. Such a lookup table thatcan be included as part of a software program, can be used and adaptedin a variety of ways. For example, if the reading is 3000 at the 20^(th)second multiply by 3, if 5000 then by 5, if 8000 then by 7. In anotherexample a set equation is used.

Conversion of counts per second to counts per minute can be accomplishedby multiplying each 1 second count by 60. In particular embodiments theluminometer, or other reader, is adjusted by reference to a standard.For example, adjusting luminometers by reference to a controlluminometer standardizes readings from one luminometer to another for agiven set of reagents and a given sample. Correction factors are used tomake the adjustments. Examples of corrections factor amount are betweenabout 200 and about 300. A formula used to calculate the correctionfactor can be:

cpm*10/correction factor,

where cpm is counts per minute calculated from a counts per secondreading. The correction factor is determined by results from aparticular luminometer with reference to a control luminometer.

In addition to the various luciferin, luciferase, buffers, detergentsand other reagents described herein, it is possible to use the methodsand devices for counting and extrapolating described herein with otherreagent systems, particularly those for measuring and detecting ATP. Invarious embodiments, reagents allowing regeneration of ATP, such asdescribed by Foote et al, U.S. Pat. No. 6,043,047, issued Mar. 28, 2000and regeneration of luciferin such as described by Kurosawa et al, EP 1306 435 A1, published May 2, 2003, may be used.

It will be appreciated to those skilled in the art that the variousherein described methods of reading and calculating a hygiene testresult, such as using an extended or cumulative count alone or inconjunction with regression analysis, and thereby detecting surfaceresidue contamination, are not limited to detection of ATP usingluciferin-luciferase based luminescence detection. For example certainof these methods may be usefully applied to reading and analyzing theresults from color based tests, for example color tests for detection ofprotein, glucose or other carbohydrates or phosphates such as thosedescribed in U.S. patent application Ser. No. 10/343,582 (HygieneMonitoring), Jan. 31, 2003 which is incorporated herein by reference.

Other examples of tests for which the herein described method of readingand extrapolating (predicting) a hygiene test result may be used, whichmay not involve ATP detection or luminescence output, include thosedescribed in U.S. Pat. No. 6,551,834, Issued Apr. 22, 2003 (Detection ofContaminants Using Self-Contained Devices Employing Target MaterialBinding Dyes); U.S. Pat. No. 6,387,650, Issued May 14, 2002 (Method andComposition for Detecting Bacterial Contamination in Food Products);U.S. Pat. No. 6,043,047, issued Mar. 28, 2000 (Sample-Collecting andAssay Device For Use In the Detection of Biological Material) andEuropean Patent 0 695 363 B1, Sep. 17, 1997 (Detection of BiologicalMaterial).

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the format of the POCKETSWAB-PLUS type device (theformat of the POCKETSWAB Ultra/H2O Plus/ALLERGIENE test device isbasically the same with only a change in the BAR solution as describedherein). FIG. 1A is a schematic view of the swab removed form the testdevice and FIG. 1B is a schematic view of the test device containing theswab. In use of the swab type device, the swab 1 is removed from thebody 3, by gripping the swab handle 2, and a 4″×4″ surface, for examplea food contact surface, is swabbed using the pre-moistened swab 1. (Theswab 1 is provided pre-moistened, for example, with the same BARsolution provided in the niblet 5.) Alternatively the swab 1 can bedipped into the sample or the sample can be pipetted onto the swab 1.The swab 1 is then reinserted into the body 3 and screwed longitudinallythrough the covering 9 of the microtube test unit 4 and through thecovering 10 of the niblet 5 and into bottom of the microtube test unit4. In an embodiment, the liquid reagent niblet 5 contains, a BARsolution such as those described herein. The liquid within the BARsolution niblet 5 dissolves the reagent 7 containingluciferin-luciferase. The resulting luminescence is read using aluminometer. Alternatively the luciferin and luciferase reagents can bein the niblet 5 with the BAR solution in the bottom of the microtubetest unit 4.

FIGS. 2A and 2B illustrate insertion of the POCKETSWAB PLUS TEST device,or POCKETSWAB Ultra/H2O Plus/ALLERGIENE Test Device into a CharmLUMINATOR-K 11 and Charm LUMINATOR-T 12). Results are read using, forexample, one of the methods described elsewhere herein, and a result isprovided on the display 13, 14. The result can be provided, for examplenumerically or, alternatively, as a positive or negative.

FIG. 3 is a graphical representation of data generated using variousconcentrations of ATP represented in the graph legend as femtomoles ATP.BAR solution contained 99.995% Butterfield's Buffer with 0.005%benzalkonium chloride. Light output was read using a LUMINATOR-T. Threehundred accumulated count per second/second readings (5 minutes total)were used to generate each line on the graph. This graph illustratesthat a cumulative reading over an extended period of time, as opposed toan average or a reading of the amplitude (peak), allows the user todistinguish lower levels of ATP.

FIG. 4 is a graphical representation of a subset of data from FIG. 3.The scale of the graph, from 0 to 1000 RLU's, further illustrates assaysensitivity relative to relatively low levels (0.01 fmol and 0.001 fmol)of ATP.

FIGS. 5A, 5B, 5C, and 5D are graphical representations of data generatedby four different types of regression analysis: power curve fit—FIG. 5A;linear trend fit—FIG. 5B; exponential curve fit—FIG. 5C; and polynomialcurve fit—FIG. 5D.

The results shown in FIGS. 6 and 7 (power curve), 8 and 9 (exponentialcurve), 10 and 11 (linear trendline), 12 and 13 (polynomial curve), and14 and 15 (average of exponential and power curves) were generated usingtwo different LUMINATOR-T analyzers. Results were generated using fivedifferent concentrations of ATP (including zero). Correction factorswere used to standardize results from both analyzers to within 5% of acontrol unit. The average of the corrected values was used. Average ofmultiple results for 20 second cumulative counts were compared to theaverage of multiple results from 300 second cumulative counts each fortwo separate LUMINATOR-T units. For each of FIGS. 6 through 15, column 1(Col 1) shows the results of 20 cps (counts per second) counts summed at20 seconds. For each of FIGS. 6 through 15, column 2 (Col 2) shows theresults of the conversion of cps to counts per minute (cpm) bymultiplying Col 1 times 60. For each of FIGS. 6 through 15, column 3(Col 3) shows the corrected value which is calculated by multiplying by10 the Col 2 results and dividing the product by the correction factor(CF) for the particular LUMINATOR-T used. For each of FIGS. 6 through15, column 4 (Col 4) shows the actual relative light unit (RLU) resultsgenerated by summing 300 cps counts after 300 seconds.

The average results were subjected to various types of regressionanalysis to determine a best fit formula for each type of regressionanalysis. Column 5 (Col 5) in FIGS. 6 and 7 (Power Curve Analysis) usedthe regression formula y=1.2539*x̂(1.183), to use 20 second results tocalculate 300 second results: R=0.99914. Column 5 (Col 5) in FIGS. 8 and9 (Exponential Curve Analysis) used the regression formulay=59597*ê(3.3426E-05x), to use 20 second results to calculate 300 secondresults: R=0.99357. Column 5 (Col 5) in FIGS. 10 and 11 (Linear CurveAnalysis) used the regression formula y=−60594+11.261x, to use 20 secondresults to calculate 300 second results: R=0.99805. Column 5 (Col 5) inFIGS. 12 and 13 (Polynomial Curve Analysis) used the regression formulay=−13737+7.5027*x+3.9039E-05x̂2: to use 20 second results to calculate300 second results: R=0.99909. Column 5 (Col 5) in FIGS. 14 and 15(Average of Exponential and Power Curves) used the regression formulay=[(59597*ê(3.3426E-05x))+(1.2539*x̂(1.183))]/2 to use 20 second resultsto calculate 300 second results. The results show that one or more ofthe best fit formulas, alone or in combination, have R valuessufficiently close to 1.0 to be able to accurately predict 5 minutecumulative results from actual 20 second cumulative results. Column 6(Col 6) shows the percentage change of regression formula predictedresults (Col 4) from actual 300 second results (Col 5).

It will be appreciated by those skilled in the art that 20 seconds and 5minutes are representative time frames and are not provided aslimitations on the scope of the invention.

FIG. 16 is a flow chart showing an example of an accumulative algorithmto increase ATP sensitivity of the Charm Luminometer. The flow chartdemonstrates obtaining a 20 second result and using that 20 secondresult to compute a final result (predicted 5 minute result). Terms usedin the flow chart include:

1) Correction—a value determined by a calibration technician to slopethe result to match a pre-calibrated standard Luminometer.2) Background—a value determined by a calibration technician to set athreshold at which detection of a contaminant is obtained and begins toshow a positive result.

3) RLU—Relative Light Units. 4) CPS—Counts Per Second.

5) Total CPM—total of CPS counts multiplied by 60 to predict TotalCounts per minute.6) Exp(x)—a Math library function which returns the exponential value ofx.7) nl(x)—a Math Library function which returns the natural logarithm ofx (base e=2.718282).

The algorithm can be within a program internal to the luminometer or inan external component to which data is transferred, such as anassociated central processing unit. The particular algorithm/flowchartshown in FIG. 16 provides a method for reading luminescent test resultsthat includes a series of count per second readings. In this algorithmthe initial count is discarded to avoid the possible impact of aninitial aberrant result. An internal temperature check can be includedto avoid incorrect results caused by excessive luminometer temperatures.Twenty separate CPS iterations are determined and summed. The total sumof the twenty CPS's can then be multiplied by 60 to arrive at a relativeCPM value. The RLU result can be determined by multiplying the CPM valueby a factor of 10 and dividing by Correction. The Correction having beendetermined by calibration to provide for consistent readings frominstrument to instrument. That corrected number can then be reduced bythe Background to arrive at the Result. That Result can then be used toextrapolate a predicted Final Result after an extended count, in thisexample using the power curve formula described.

Another useful algorithm is shown in FIGS. 17A and 17B. In thisalgorithm the initial one second count is discarded to avoid thepossible impact of an initial aberrant result. This step can be repeatedmultiple times, for a pre-determined Delay Time, to allow additionalreagent and instrument stabilization, if necessary. In this algorithm avisual indication of result is provided after each RLU calculation sothat the user can observe results after each count summation andcalculation and, therefore, terminate the test earlier if a positiveresult is established. The total sum of the twenty CPS's can then bemultiplied by 600 and divided by the Correction to simplify theequation. In this example, as distinguished from the example provided inFIG. 16, Background counts are deducted from the Final Result after theextended count prediction formula is applied.

EXAMPLES Example 1

BAR solution containing 99.995% modified Butterfield's Buffer (made withdibasic rather than monobasic phosphate) with 0.005% benzalkoniumchloride was prepared as follows:

A. Prepare 1 L of 0.195 M dibasic potassium phosphate as follows:

-   1. Add 34 g dibasic potassium phosphate (K₂HPO₄; FW=174.2) to 500 mL    ultra-pure water in a 1 liter Erlenmeyer flask. Mix until fully    dissolved.-   2. Adjust pH to 7.20±0.02 with phosphoric acid.-   3. Bring volume to 1 L with ultra-pure water. Mix for 5 minutes.    B. Prepare 22 L of BAR solution (0.24 mM dibasic potassium    phosphate) as follows:-   1. Add 27.5 mL of 0.195 M dibasic potassium phosphate to carboy with    mark at 22 L.-   2. Bring volume to 22 L with ultra-pure water. Mix for 5 minutes.-   3. Dispense 3500 mL of BAR solution into six×4 L NALGENE (Nalgene is    a registered trademark of Nalge Nunce International Corporation    Rochester, N.Y.)    C. Autoclave the bottles of BAR solution for 90 minutes at 121° C.    D. After autoclaving add 350 μL of 50% benzalkonium chloride to each    bottle of BAR solution once the solution has cooled to room    temperature. Shake well for 30 seconds to mix.

The above described BAR solution can be used with luciferin-luciferasereagents to detect ATP. In one example luciferin-luciferase wereproduced using highly purified beetle D-Luciferin free acid from RegisTechnologies, Inc., Catalog # 360100; and r-luciferase was from PROMEGA,specific activity 3.3×10¹⁰ relative light units per mg protein (minimumspecification 2.0×10¹⁰ relative light units per mg protein). Theluciferin and luciferase were freeze dried together with magnesiumacetate and ATP-free bulking and stabilizing agents such as lactose,BSA, glycine (1.4 millimolar glycine when combined with 300 microliterBAR solution), ethylenediaminetetraacetic Acid (EDTA), dithiothreitol(DTT), and stabilizers such as lactose, and tableted after addition ofAVICEL and magnesium stearate. The ratio of luciferin to BAR solutionwas about 0.073 micrograms luciferin per microliter BAR solution. Theratio of luciferase to BAR solution ratio was about 0.0073 microgramsluciferase per microliter buffer.

Example 2

For testing, 300 μL of BAR solution was used (various compositionsdescribed below) and either luciferin-luciferase liquid solution, withmagnesium co-factor or without magnesium cofactor. Tabletedluciferin-luciferase (which contains additional stabilizers andmagnesium cofactor) was also tested for comparison. In either case, theamount of luciferin per test was 23 μg, and the amount of luciferase was2.3 μg. Luciferin/Luciferase tablet includes co-factor, stabilizers andbulking agents including: magnesium acetate, lactose, BSA, glycine,EDTA, DTT, lactose, AVICEL (AVICEL is a registered trademark of FMCCorporation, Philadelphia, Pa.) and magnesium stearate.Luciferin/Luciferase liquid includes only luciferin and luciferase oralternatively as indicated luciferin and luciferase and magnesium. LUM-Tbackground was set to 100 (“off”) in order to see differences inuncorrected background values. Results presented in Table 1 show that ofthe various niblet solutions tested those with Butterfield's Buffer and0.005% benzalkonium chloride had the best positive to negative ratio.The niblet solution including 260 mM Tris base/75 mM tricine, pH 7.8 isused in the niblet solution for the “standard” POCKETSWAB PLUS (CharmSciences, Inc. Lawrence, Mass.). These results show maximum sensitivityusing a BAR solution of BB+BC as compared with other possible BARsolutions. In table 1 the abbreviation BB stands for Butterfield'sBuffer prepared with dibasic potassium phosphate and the abbreviation BCstands for benzalkonium chloride.

TABLE 1 0.01 fmol Niblet Solution Luciferin- Negative ATP Pos/NegComposition Luciferase RLU Avg RLU Avg Ratio Control (BB 99.995% +tablet 55746 190453 3.42 BC 0.005%) Sterile water tablet 57571 987421.72 BB alone (no BC) tablet 47199 142736 3.02 260 mM Tris base/75tablet 48869 66665 1.36 mM Tricine; pH 7.8 20 mM Tris base/ tablet 75869151421 2.00 5 mM Tricine; pH 7.8 Control (BB 99.995% + liquid 3759640595 1.08 BC 0.005%) Sterile water liquid 33300 32186 0.97 Control (BB99.995% + Liquid + 53230 169660 3.19 BC 0.005%) Mg

Example 3

An embodiment involves detection of bacterial contamination in water,for example food production water, rinse water or wet surfaces fromcleaned equipment. In this embodiment a sample swab is dipped in samplewater and swirled for approximately 5 seconds. After removal from thewater sample, the swab is contacted with BAR solution, for example BARsolution containing 99.995% dibasic phosphate buffer and 0.005%benzalkonium chloride. The phosphate buffer has molarity in the range ofabout 0.1 millimolar to about 0.5 millimolar. After adding sample to theBAR solution the mixture is contacted with the luciferin/luciferasereagents for example the tablet described with reference to Example 1.Luminescence results are determined on a standard luminometer, forexample a LUMINATOR-T, FIREFLY, LUMGIENE or NOVALUM (Charm Sciences,Inc.), utilizing a 20 second cumulative count and Power Curve generatedresult.

Examples 4-7

NOTE: The following examples 4-7 and related tables show detectionlevels for common food residues, for example 5 ppm peanut butter.Generally, the allergenic component of the food, such as peanut butter,is a protein. As a result, detection levels may refer to peanut butterprotein levels rather than peanut butter levels. If peanut butter isdetected at 5 ppm and peanut butter is, generally, about 22% proteinthen peanut butter protein can be detectable at 5 ppm×0.22, orapproximately 1.1 ppm protein.

Example 4

Table 2 shows results using a BAR solution of 99.995% dibasic phosphatebuffer and 0.005% benzalkonium chloride to test various potentiallyallergenic matrices (unsalted cocktail peanuts, peanut butter,pasteurized whole milk, raw egg white, raw whole egg, all-purpose flour)at a variety of concentrations. A 5000 ppm stock solution of each foodwas prepared in sterile water, then diluted serially in sterile water.Results were generated on a luminometer utilizing a 30 second cumulativeRLU count and a background subtract of 15000. For testing, a 20microliter sample was added directly to a swab. A LUMINATOR-K was used.

Luciferin-luciferase was as described in Example 1. The results show ATPdetection at levels at which a positive result can be used to determine,for example, that peanut butter may be present at above 5 ppm.

TABLE 2 Allergen/Source RLU Cumulative % Positive Blank (negativecontrol) 0 0% Unsalted cocktail Peanut peanuts (25% protein) 5000 ppm711557 100% 500 ppm 65217 100% 50 ppm 8247 100% 25 ppm 6630 100% 5 ppm 00% Peanut butter Peanut Butter (22% protein) 5000 ppm 957200 100% 500ppm 101881 100% 50 ppm 7851 100% 25 ppm 7072 100% 5 ppm 1850 33%Pasteurized whole milk Milk (~2.7% casein) 18500 ppm 2447813 100% 1850ppm 284021 100% 185 ppm 19942 100% 37 ppm 1773 50% Raw egg white EggWhite (~10% protein) 50000 ppm 20263 100% 25000 ppm 11270 100% 10000 ppm768 100% 5000 ppm 2519 50% Raw whole egg 5000 ppm 72812 100% (~6.7% eggwhite 500 ppm 16578 100% protein) 250 ppm 4939 100% 50 ppm 0 0%All-purpose flour Flour (~10% protein) 5000 ppm 266208 100% 500 ppm34620 100% 250 ppm 34188 100% 100 ppm 1780 100% 50 ppm 0 0%

Example 5

Table 3 shows results using a BAR solution of 99.995% dibasic phosphatebuffer and 0.005% benzalkonium chloride with luciferin/luciferasedescribed in Example 1, to test the same allergenic matrices as Example4. A 5000 ppm stock solution of each food was prepared in sterile water,then diluted serially in sterile water. A 5 second average RLU(non-cumulative in which five RLU counts per second over five secondsare averaged) count and a background subtract of 2600 were used. Fortesting, a 20 microliter sample was added directly to a swab. ALUMINATOR-K was used. Sensitivity decreased as compared to results shownin Example 4.

TABLE 3 Allergen Source RLU % Positive Blank (negative control) 0 0%Unsalted cocktail peanuts (25% Peanut protein) 5000 ppm 16575 100% 500ppm 679 50% 50 ppm 0 0% 25 ppm 0 0% 5 ppm 0 0% Peanut butter (22%protein) Peanut Butter 5000 ppm 22264 100% 500 ppm 1000 50% 50 ppm 0 0%25 ppm 0 0% 5 ppm 0 0% Pasteurized whole milk (~2.7% Milk casein) 18500ppm 62521 100% 1850 ppm 5687 100% 185 ppm 0 0% 37 ppm 0 0% Raw egg white(~10% protein) Egg White 50000 ppm 0 0% 25000 ppm 0 0% 10000 ppm 0 0%5000 ppm 0 0% Raw whole egg (~6.7% egg white Whole Egg protein) 5000 ppm0 0% 500 ppm 0 0% 250 ppm 0 0% 50 ppm 0 0% All-purpose flour (~10%protein) Flour 5000 ppm 4663 100% 500 ppm 0 0% 250 ppm 0 0% 100 ppm 0 0%50 ppm 0 0%

Example 6

Table 4 shows results using BAR solution of 260 mM Tris base/75 mMtricine, pH 7.8 (as previously used in the POCKETSWAB-PLUS test).Luciferin-luciferase described in Example 1. The same allergenicmatrices were tested as in Example 4. A 5000 ppm stock solution of eachfood was prepared in sterile water, then diluted serially in sterilewater. Results were generated on a luminometer utilizing a 5 secondaverage RLU (non-cumulative in which five RLU counts per second overfive seconds are averaged) count and a background subtract of 2600. Fortesting, a 20 microliter sample was added directly to a swab. ALUMINATOR-K was used. Results show decreased sensitivity as comparedwith results in both Example 4 and Example 5.

TABLE 4 Allergen Source RLU % Positive Blank (negative control) 0 0%Unsalted cocktail peanuts (25% Peanut protein) 5000 ppm 1916 100% 500ppm 0 0% 50 ppm 0 0% 25 ppm 0 0% 5 ppm 0 0% Peanut butter (22% protein)Peanut Butter 5000 ppm 4399 100% 500 ppm 0 0% 50 ppm 0 0% 25 ppm 0 0% 5ppm 0 0% Pasteurized whole milk (~2.7% Milk casein) 18500 ppm 18473 100%1850 ppm 2276 100% 185 ppm 0 0% 37 ppm 0 0% Raw egg white (~10% protein)Egg White 50000 ppm 0 0% 25000 ppm 0 0% 10000 ppm 0 0% 5000 ppm 0 0% Rawwhole egg (~6.7% egg white Whole Egg protein) 5000 ppm 0 0% 500 ppm 0 0%250 ppm 0 0% 50 ppm 0 0% All-purpose flour (~10% protein) Flour 5000 ppm0 0% 500 ppm 0 0% 250 ppm 0 0% 100 ppm 0 0% 50 ppm 0 0%

Example 7

Table 5 shows results using a BAR solution of 99.995% dibasic phosphatebuffer and 0.005% benzalkonium chloride with luciferin/luciferasedescribed in Example 1, to test various concentrations of potentiallyallergenic matrices. A 5000 ppm stock solution of each food was preparedin sterile water, then diluted serially in sterile water. A 5 minutecumulative count was used. Background subtract of 100,000 was used. ALUMINATOR-K was used. Increased sensitivity, as compared with resultsfrom the next most sensitive combination (results in Example 4) wasobserved.

Further experiments, using a 10 minute cumulative count, increasedsensitivity to egg white to 5 ppm (approximate egg white proteinequivalent of 0.5 ppm). It is expected that sensitivity to othermatrices may also be increased using such an extended cumulative count.

TABLE 5 Food Concentration RLU Average % Positive Allergen TestManufacturer Claims Peanut butter (22% protein) 5 ppm 747242 100% <0.1to 1 ppm 0.5 ppm 83137 100% peanut protein 0.25 ppm 60753 100% (0.5 to 3hr assay time) 0.1 ppm 59232  67% 0.05 ppm 5188  33% Soy nuts (36.7%protein) 50 ppm 199083 100% 70 to <5000 ppm 5 ppm 61132 100% soy protein0.5 ppm 9414 100% (30 min assay time) 0.05 ppm 0  0% Almond (20%protein) 50 ppm 217604 100% 1.7 to 5 ppm 5 ppm 71092 100% almond 0.5 ppm4396  50% (30 min assay time) Walnut (16.7% protein) 50 ppm 182168 100%unknown 5 ppm 51090 100% 0.5 ppm 225  50% Pecan (10% protein) 50 ppm76873 100% unknown 5 ppm 10257 100% 0.5 ppm 0  0% Egg white (10%protein) 5000 ppm 224516 100% 1 to 5 ppm egg 500 ppm 40575 100% whiteprotein 100 ppm 33374 100% (0.5 to 1.5 hr assay time) 50 ppm 7860  67%Whole egg (6.7% protein) 500 ppm 41404 100% 1 to 5 ppm egg 50 ppm 40977100% white protein 5 ppm 6809  67% (0.5 to 1.5 hr assay time) 0.5 ppm187  33% Whole milk, pasteurized (2.7% casein) 100 ppm 223233 100% 5 ppm10 ppm 105121 100% milk protein 1 ppm 29771  67% (0.5 to 2 hr assaytime) Whole wheat four (13.3% protein) 100 ppm 440020 100% <2 to 8 ppm10 ppm 77042 100% wheat proteins 1 ppm 26010 100% (0.5 to 2 hr assaytime) 0.1 ppm 32217  67% All-purpose white four (10% protein) 10 ppm82308 100% <2 to 8 ppm 1 ppm 29483 100% wheat proteins 0.1 ppm 0  0%(0.5 to 2 hr assay time) Clams, raw (12.8% protein) 50 ppm 130018 100%unknown 25 ppm 55383 100% 10 ppm 28197  40% 5 ppm 0  0% Shrimp, raw(20.3% protein) 50 ppm 129186 100% unknown 25 ppm 82211 100% 10 ppm39574  80% 5 ppm 1613  33% Atlantic salmon, raw (19.9% protein) 50 ppm48824 100% unknown 25 ppm 45425 100% 10 ppm 8237  60% 5 ppm 0  0%Soybeans (36.5% protein) 50 ppm 346842 100% 70 to <5000 ppm 5 ppm 30648100% soy protein 0.5 ppm 3878  50% (30 min assay time) Sunflower seeds(20% protein) 5 ppm 93950 100% unknown 0.5 ppm 42704 100% 0.05 ppm 33843 40% Sesame seeds (17.7% protein) 500 ppm 508324 100% 1 ppm sesame 50ppm 38680 100% seed protein 25 ppm 8551  60% (assay time unknown) Wholemilk, powdered (19.7% casein) 50 ppm 399603 100% 5 ppm 5 ppm 50471 100%milk protein 0.5 ppm 16748  40% (0.5 to 2 hr assay time) Soy flour (52%protein) 50 ppm 399366 100% 70 to <5000 ppm 5 ppm 95755 100% soy protein0.5 ppm 31848  60% (30 min assay time) Whole milk, UHT (2.7% casein)*1000 ppm 32680 100% 5 ppm 500 ppm 30557 100% milk protein 250 ppm 10541 50% (0.5 to 2 hr assay time) 100 ppm 2354  20%

Example 8

Tables 6, 7, 8, and 9 show results using using a BAR solution of 99.995%dibasic phosphate buffer and 0.005% benzalkonium chloride withluciferin/luciferase described in Example 1, to test variousconcentrations of ATP (Table 6) Whole Egg (Table 7), Peanut Butter(Table 8) and Egg White (Table 9). RLU counts were read every second for300 seconds and totaled. Background subtract of 100,000 was used.Results in tables 6, 7, 8 and 9 show multiple RLU readings which wereaveraged to arrive at particular results. These results are alsosummarized in particular examples in Example 7 (Table 5). A LUMINATOR-Kwas used to detect the luminescence.

TABLE 6 ATP Concentration (fmol/20 uL sample) RLU Readings % Positive0.5 4196324 100% 6406563 5501847 Avg: 5368245 0.1 4408690 100% 21389192345606 Avg: 2964405 0.01 213632 100% 250782 195101 Avg: 219838  0.00199087 100% 37798 40974 Avg: 59286  0% 0  0% 0 0 Avg: 0   

TABLE 7 Whole Egg Concentration (ppm) RLU Readings % Positive 500 31407100% 11446 81360 Avg: 41404 50 2715 100% 100510 19707 Avg: 40977 5 1851667% 1911 0 Avg: 6809  0.5 562 33% 0 0 Avg: 187  0 0 0% 0 0 Avg: 0  

TABLE 8 Peanut Butter Concentration (ppm) RLU Readings % Positive 5889650 100% 628410 723666  Avg: 747242 0.5 135814 100% 54646 58950 Avg:83137 0.25 3473 100% 42586 136200 Avg: 60753 0.1 116374 67% 61321 0 Avg:59232 0.05 0 33% 0 15564 Avg: 5188  0 0 0% 0 0 Avg: 0  

TABLE 9 Egg White Concentration (ppm) RLU Readings % Positive 5000 (500ppm protein) 147025 100% 302007  Avg: 224516 500 (50 ppm protein) 47696100% 35638 38391 Avg: 40575 100 (10 ppm protein) 29355 67% 15956 54812Avg: 33374 50 (5 ppm protein) 12769 67% 0 10810 Avg: 7860  0 0 0% 0 0Avg: 0  

Example 9

Tables 10-13 show a comparison of RLU results using three different BARsolutions (referred to as BAR A, BAR B and BAR C). Luciferin/luciferasewas described in Example 1. BAR A solution (original formulation fromPocketSwab Plus swabs) contained 3.138% Trizma Base, 3.125% phosphoricacid detergent, 1.344% Tricine, 1.344% Triton X-100 (10% solution) and0.172% benzalkonium chloride (10% solution) and deionized water.Displacement measurements for Trizma base and tricine were used tocalculate the volume of deionized water needed. BAR B solution containedButterfield's Buffer made with dibasic phosphate (less than about 1millimolar). BAR C solution contained 99.995% Butterfield's Buffer madewith dibasic phosphate (less than about 1 millimolar) with 0.005%benzalkonium chloride.

Table 10 shows the average RLU results from a comparison of BAR A, BAR Band BAR C using the luciferin/luciferase described with reference toExample 1 and Example 1 and varying concentrations of ATP from 0 to 180femtomoles. Both the LUMT and FIREFLY luminometers were used withbackground subtract. Results show increased sensitivity by decreasingthe molarity of the BAR solution, using only Butterfield's Buffer (lowmolarity phosphate buffer) made with dibasic phosphate. BAR C shows thebest sensitivity when 0.005% benzalkonium chloride is added toButterfield's Buffer made with dibasic phosphate.

TABLE 10 LUMT FIREFLY BAR-A 81,900 21,013 180 fmoles ATP BAR-B 129,93334,459 BAR-C 218,366 65,112 BAR-A 8,368 752 18 fmoles ATP BAR-B 9,7481,066 BAR-C 16,636 2,665 BAR-A 0 0 1.8 fmoles ATP BAR-B 0 0 BAR-C 2,8690 BAR-A 0 0 0 fmoles ATP BAR-B 0 0 BAR-C 0 0

Table 11 shows comparative RLU results using BAR A, BAR B and BAR C andsample uptake by swabbing a surface contaminated with a variety of foodresidues. The results again show overall increased sensitivity using BARC. Surface squares tested with finished unit on LUMINATOR-T withbackground subtract.

TABLE 11 Solution Chicken Juice Egg Milk BAR-A 130582 3800 9902 4777BAR-C 169327 89186 60948 51338 BAR-B 209985 24938 23509 29156

Table 12 shows the average RLU results from a comparison of BAR A, BAR Band BAR C using the luciferin/luciferase described in Example 1 andvarying concentrations (dilutions) of a variety of bacteria by pipetting10 microliters onto each swab. LUMINATOR-T was used with backgroundsubtract.

TABLE 12 Bacterial study done by pipetting 10 ul onto each swab systemC. S. P. P. Solution freundii cerevisiae agglomerans fluorescens BAR-A10315 36163 30633 5931 Diln BAR-C 15280 582814 45942 5525 10-2 BAR-B12603 56975 18555 21306 BAR-A 0 2509 5180 0 Diln BAR-C 115 53092 1953513 10-3 BAR-B 0 3828 14375 0 BAR-A 0 0 8646 0 Diln BAR-C 0 1556 275 010-4 BAR-B 0 3868 3880 0

Tables 13A, 13B, 13C, 13D, 13E, 13F show results from a variety ofstability, sensitivity and test background experiments.

Table 13A—The results show increased count stability between 1 and 2minutes after test initiation using a 180 femtomole concentration of ATPand BAR A versus BAR C. Results shown are from the same test recountedand show that BAR C, in addition to increased sensitivity, providedincreased test result stability. LUMINATOR-T with background subtract.

180 fmoles Initial 1 min % change 2 min % change BAR-A 111,065 94,115−15% 70,426 −37% BAR-C 205,833 205,638  −0.10%   196,642   −4%

Table 13B—The results show a comparison of results using BAR B and BAR Ctesting a 60 femtomole concentration of ATP. LUMINATOR-K was used withno background subtract.

Zero 60 fmoles ATP BAR-B 450 136,000 BAR-C 650 242,700

Table 13C—The results show count stability between an initial count anda one minute count, comparing BAR A and BAR C. LUMINATOR-T used withbackground subtract.

60 fmoles ATP Initial Count Count after 1 min. BAR-A 30,581 17,049 BAR-C252,365 240,069

Table 13D—The results show count stability between an initial count anda one minute count, comparing BAR A and BAR C in testing a 10 microlitersample of raw milk pipetted onto a swab and then the swab is contactedwith the BAR solution. LUMINATOR-T used with background subtract.

Initial count Count after 1 min. BAR-A 115,459 223,941 BAR-C 501,329720,153

Table 13E—The results show temperature stability of BAR C in temperaturestressed conditions versus standard conditions. LUMINATOR-K used with nobackground subtract.

0 count 3.6 fmoles ATP BAR-C 661 14,644 BAR-C (stressed) 711 15,173

Table 13F—The results show increased sensitivity to 1.8 femtomoles ATPusing BAR C as compared to BAR B and BAR A. LUMINATOR-T used withbackground subtract.

Zero check 1.8 fmoles ATP BAR-A 0, 0, 0, 0, 0 0, 0 BAR-C 0, 0, 0, 0, 03928/3236 BAR-B 0, 0, 0, 0, 0 828/567

Example 10

Table 14 shows RLU results using BAR solution of varying millimolarconcentrations of Bis-Tris Propane (1 mM-250 mM) pH 6.6 with 0.05%Triton X-100, 0.02% benzalkonium chloride and 10 mM magnesium acetate.The Bis-Tris results are compared with the control of Butterfield'sBuffer (0.31 mM monobasic potassium phosphate, pH 7.2) and 0.005%benzalkonium chloride. The signal to noise ratio (positiveresult/negative result ratio, provided in the parenthesis) of the 50 mMformulation results compared favorably to signal to noise ratio of the 1mM results and the control. The 50 mM buffer, however, can be moredesirable in that it provides better pH stabilization.

TABLE 14 Buffer Zero 0.05 fmol ATP 0.01 fmol ATP Control 53248 269829(5.1) 149186 (2.8) 250 mM 24659  40448 (1.6)  31084 (1.3) 100 mM 31808160852 (5.1)  63116 (2.0) 50 mM 46604 266492 (5.7) 140848 (3.0) 10 mM64707  377231 (5..8) 187116 (2.9) 1 mM 92904 505437 (5.4) 281260 (3.0)

Example 11

Table 15 compares results using the optimum buffer concentration fromtable 14 (50 mM Bis-Tris Propane (BTP) without surfactants, with 10 mMmagnesium acetate, and with recombinant luciferase from PROMEGA at avariety of pH's from pH 6.0 to 9.5. 300 mL of buffer was used with 2.3uL of 10 mg/mL luciferin and 2.3 uL of 1 mg/mL luciferase. At each pH,both zero results and 0.01 fmol (of ATP-20 uL solution) results are theaverage of 3 readings. Results show that pH 6.5 provides the optimumsignal to noise ratio. Results also show that the signal to noise ratio,using recombinant luciferase, can be improved without maximizingluminescence output by providing a decreased background (noise).

TABLE 15 pH 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Zero 24468 41531 170450240434 216444 125177 114931 40990 0.01 fmol 33568 144012 248227 451251298006 203672 161284 37106 Pos/Neg ratio 1.4 3.5 1.5 1.9 1.4 1.6 1.4 0.9

Table 16 compares results using the optimum formulation from table 14(50 mM Bis-Tris Propane (BTP), without surfactants and with 10 mMmagnesium acetate, with natural luciferase at a pH range of pH 6.0 to pH9.5. 300 mL of buffer was used with 2.3 uL 10 mg/mL luciferin and 2.3 uL1 mg/mL luciferase. At each pH, both zero results and 0.01 fmol (ofATP-20 uL solution) results are the average of 3 readings. Results showthat less sensitivity as compared to recombinant luciferase and that,using natural luciferase, at pH 6.5 did not provide as much sensitivityas pH 7.5 and that the maximum signal to noise ratio was achieved at apH were the luminescence was maximized.

TABLE 16 pH 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Zero 28970 56213 185104205232 217513 133778 53763 20586 0.01 fmol 27080 82236 242685 379648310669 147994 70699 23529 Pos:neg ratio 0.9 1.5 1.3 1.8 1.4 1.1 1.3 1.1

Table 17 compares results using the optimum formulation from table 14(50 mM Bis-Tris Propane (BTP), without surfactants and with 10 mMmagnesium acetate, with recombinant luciferase from PROMEGA at a pHrange from pH 6.0 to pH 7.0. 300 mL of buffer was used with 2.3 uL of 10mg/mL luciferin and 2.3 uL of 1 mg/mL luciferase. At each pH, both zeroresults and 0.01 fmol (of ATP-20 uL solution) results are the average of3 readings. Results indicate that at pH 6.5 and pH 6.6 results showedthe largest signal to noise ratio.

TABLE 17 pH 6.0 6.1 6.2 6.3 6.4 6.5 Zero 56656 28352 32525 38976 4639051708 0.01 35307 40459 59495 95783 119357 156861 fmol Pos:neg 1.3 1.41.8 2.5 2.6 3.0 ratio pH 6.6 6.7 6.8 6.9 7.0 Zero 65716 102317 136208161982 200930 0.01 187778 232729 259382 357082 382279 Fmol Pos:neg 2.92.3 1.9 2.2 1.9 Ratio

Controls for comparison to tables 14-17 included:

1) 50 mM BTP, pH 6.6, with 0.05% Triton X-100, 0.02% benzalkoniumchloride. 10 mM magnesium acetate was provided in a solution of 2.3 uL10 mg/mL luciferin and 2.3 uL 1 mg/mL recombinant luciferase fromPROMEGA. Results for blanks (average of 3) were 40376 and results for0.01 fmol ATP (average of 3) were 160494, for a positive:negative ratioof 4.0. This indicated that the surfactants provided some additionalsensitivity at the apparent optimum buffer conditions;2) 50 mM BTP, pH 6.6, with 0.05% Triton X-100, 0.02% benzalkoniumchloride. 10 mM magnesium acetate was provided in a solution of 2.3 uL10 mg/mL luciferin and 2.3 uL 1 mg/mL natural luciferase. Results forblanks (average of 3) were 111227 and results for 0.01 fmol ATP (averageof 3) were 165473, for a positive:negative ratio of 1.5. This confirmedthe reduced sensitivity as compared to recombinant luciferase using theapparent optimum conditions for the recombinant reaction. The reductionin sensitivity occurring primarily from the increased negative(background) result; and3) 50 mM BTP, pH 6.6, with 0.05% Triton X-100, 0.02% benzalkoniumchloride. Luciferin/luciferase were provided with magnesium acetate in atablet, provided within POCKETSWAB PLUS (from Charm Sciences, Inc.)known as the reagent C tablet. Results for blanks (average of 3) were41222 and results for 0.01 fmol ATP (average of 3) were 164988, for apositive:negative ratio (signal to noise) of 4.0. This confirmed thatadditional reagent C tablet components were not substantiallycontributing to the increased sensitivity, as compared to the liquidformulation described above.

Example 12

To determine the optimum range of detergent concentrations varyingconcentrations of Triton X-100 were tested. Buffer was 50 mM Bis-Tris.Controls included: 50 mM Bis-Tris without detergents and 50 mM Bis-Triswith 0.05% Triton X-100 and 0.02% benzalkonium chloride. Results shownin Table 18 show that although 0.1% Triton X-100 provides the largestdifference between positive and negative, the background result (blankresult) was relatively high justifying possibly utilizing a lowerconcentration that did not provide such a large difference betweenpositive and negative but provided a lower background, in this case0.05% Triton X-100. Results are average of three tests.

TABLE 18 % Triton X-100 0 0.001 0.005 0.01 0.05 0.1 0.25 0.5 Zero 3185035786 36528 38452 48708 67671 96085 122869 0.01 fmol 102644 136453127318 140865 185585 272666 353877 392306 Pos:neg ratio 3.2 3.8 3.5 3.73.8 4.0 3.7 3.2Control results using 50 mM Bis-Tris with 0.05% Triton X-100 and 0.02%benzalkonium chloride were 44658 for negative (background) and 175659for 0.01 fmol ATP. Results were average of three results.

1. A method for sensitive detection of ATP in a sample comprising thesteps of: a) combining a sample to be tested with a solution to create afirst admixture; b) admixing said first admixture with reagents to forma second admixture, said reagents comprising luciferin and recombinantluciferase, said second admixture comprising at least one buffer whereina reaction caused by the admixing of said first admixture with saidreagents to create said second admixture generates luminescence; and c)detecting said generated luminescence as an indication of the presenceof ATP in said sample, wherein the ATP concentration of the secondadmixture is less than about 3.0 picomolar, and wherein a pH of thesecond admixture is a pH at which total generated luminescence is belowa maximum level and is a pH at which a signal to noise ratio ismaximized.
 2. The method of claim 1 wherein the ATP concentration of thesecond admixture is about 2.0 picomolar to about 3.0 picomolar.
 3. Themethod of claim 1 wherein the ATP concentration of the second admixtureis about 1.0 picomolar to about 2.0 picomolar.
 4. The method of claim 1wherein the ATP concentration of the second admixture is less than about1.0 picomolar.
 5. The method of claim 1 wherein the luciferase isexpressed using a recombinant DNA coding for Coleoptera luciferase.
 6. Amethod for sensitive detection of ATP in a sample comprising the stepsof: a) combining a sample to be tested with a solution to create a firstadmixture; b) admixing said first admixture with reagents to form asecond admixture, said reagents comprising luciferin and Coleopteraluciferase, said second admixture comprising at least one buffer whereina reaction caused by the admixing of said first admixture with saidreagents to create said second admixture generates luminescence; and c)detecting said generated luminescence as an indication of the presenceof ATP in said sample, wherein the ATP concentration of the secondadmixture is less than about 3.4 picomolar, and wherein a pH of thesecond admixture is a pH at which total generated luminescence is belowa maximum level and is a pH at which a signal to noise ratio ismaximized.
 7. The method of claim 6 wherein the ATP concentration of thesecond admixture is about 3.0 picomolar to about 3.4 picomolar.
 8. Themethod of claim 6 wherein the ATP concentration of the second admixtureis about 2.0 picomolar to about 3.0 picomolar.
 9. The method of claim 6wherein the ATP concentration of the second admixture is about 1.0picomolar to about 2.0 picomolar.
 10. The method of claim 6 wherein theATP concentration of the second admixture is less than about 1.0picomolar.
 11. The method of claim 6 wherein said solution furthercomprises at least one detergent.
 12. The method of claim 6 wherein thedetecting of generated luminescence comprises measuring sampleluminescence with a luminometer, said luminometer comprising aphotomultiplier tube, and wherein the total luminescence generated,during a predetermined period of time, is measured and used as theindication of the presence of ATP in said sample.
 13. The method ofclaim 12 wherein the detecting of generated luminescence comprisesmeasuring the total luminescence generated, during a predeterminedperiod of time, and wherein said total luminescence is used to predictthe total luminescence that would be generated over a period of timelonger than said predetermined period of time and wherein said predictedtotal luminescence is used as an indication of the presence of ATP inthe sample.
 14. The method of claim 6 wherein the luciferase isexpressed using a recombinant DNA coding for Coleoptera luciferase. 15.The method of claim 6 wherein said luciferin comprises a Coleopteraluciferin.
 16. The method of claim 6 wherein said luciferase comprises arecombinant Coleoptera luciferase and said luciferin comprises aColeoptera luciferin.
 17. The method of claim 6 wherein said Coleopteraluciferase comprises luciferase of an insect selected from the familiesLampyridae and Elateridae.
 18. The method of claim 6 wherein saidColeoptera luciferase comprises luciferase of an insect selected fromthe genus Photinus.
 19. The method of claim 6 wherein said Coleopteraluciferase comprises luciferase of the species Photinus pyralis.
 20. Themethod of claim 6 wherein said Coleoptera luciferase comprisesluciferase of an insect from the Diptera family.